Zainab Amin (IISER Pune, India)
LinkedIn: @Zainab Khan; X: @ZAINAB_KHAN_7
Abstract: HSPB8 (Heat Shock Protein B8) is an important chaperone that acts independently of ATP. Perturbations in HSPB8 function have thus been implicated in various protein aggregation disorders. Despite its biological importance, the structural and dynamic behaviour of HSPB8 under different stress conditions remains poorly understood. Understanding these perturbations is a key to elucidating the role of HSPB8 in protein quality control mechanisms. In this study, we performed a biophysical characterization of the α-crystallin domain (ACD) of HSPB8, involved in dimer formation, using solution-state nuclear magnetic resonance spectroscopy under different environmental perturbations. The effect on the structural integrity was characterized by monitoring changes in chemical shifts and linewidths of ACD in response to stressors. The results suggest that the ACD domain of HSPB8 is highly sensitive to environmental perturbations. In parallel, an initial investigation into the folding process of the protein has been carried out using multidimensional NMR spectroscopy. The backbone amide resonances of the unfolded protein were assigned through a combination of 3D NMR experiments, allowing mapping of amino acid residues to their respective peaks in the 2D 15N-1H HSQC spectrum. With the unfolded state characterized, this study aims to further elucidate the conformational landscape of the protein during refolding by gradually reducing the denaturant concentration and monitoring changes using the dynamic NMR techniques. These experiments are expected to yield mechanistic insights into the folding pathway of HSPB8, including the identification of transient, low-population intermediate states that may play critical roles in its chaperone activity and cellular function under stress.
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Thanks for a nice presentation. I have following questions regarding the same:
What happens if you go in reverse order i.e. If the protein is denatured slowly with the help of urea and HSQC is recorded? -
Thanks for asking! We have not tried that as it is difficult to assign the protein its folded monomeric form as of now. I think the protein may or may not follow the same folding pathway as we slowly unfold the protein from the folded form. To have an exact answer ,we may need to perform the experiments.
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Thank you for this nice presentation!
Your CEST profiles are quite pretty. I was wondering though if you had any information about the intermediate state, i.e. what is its nature? I thought that if it is was a folding intermediate, its chemical shift would be closer to the folded state but it seems like the major peak is moving away as you decrease the urea concentration.
In addition, as you progress toward folding, do you expect to form oligomeric species? How do the R2 of the ground and excited states compare?-
The observation that the chemical shifts of the minor (excited) state are distinct from both the unfolded and native conformations suggests that the intermediate represents a unique conformational ensemble. While it may involve local structure formation, it remains structurally distinct from the final folded state, a point further supported by our HSQC spectra. Although the fully folded state has not yet been assigned, overlay analysis shows that the intermediate does not fully converge with it, particularly at 2 M urea, where the HSQC profile deviates from the native-like pattern. The minor-state chemical shifts remain relatively consistent across decreasing urea concentrations, which indicates that the intermediate is structurally persistent. Regarding oligomerization, our concentration-dependent HSQC experiments for the folded construct showed only subtle line shape changes, consistent with weak self-association. This suggests a tendency towards dimer formation, though not strong enough to classify as higher-order oligomerization under the conditions tested. At 2 M urea, we do observe increased R₂ values and modest peak broadening, yet not to the extent typically seen with large oligomeric assemblies. Further validation is required to confirm this behavior.
I hope this helps clarify some of your questions!
Thank you!
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The observation that the chemical shifts of the minor (excited) state are distinct from both the unfolded and native conformations suggests that the intermediate represents a unique conformational ensemble. While it may involve local structure formation, it remains structurally distinct from the final folded state, a point further supported by our HSQC spectra. Although the fully folded state has not yet been assigned, overlay analysis shows that the intermediate does not fully converge with it, particularly at 2 M urea, where the HSQC profile deviates from the native-like pattern. The minor-state chemical shifts remain relatively consistent across decreasing urea concentrations, which indicates that the intermediate is structurally persistent. Regarding oligomerization, our concentration-dependent HSQC experiments for the folded construct showed only subtle line shape changes, consistent with weak self-association. This suggests a tendency towards dimer formation, though not strong enough to classify as higher-order oligomerization under the conditions tested. At 2 M urea, we do observe increased R₂ values and modest peak broadening, yet not to the extent typically seen with large oligomeric assemblies. Further validation is required to confirm this behavior.
I hope this helps clarify some of your questions!
Thank you! -
Hi Zainab, nice talk! I am curious about how you can use this technique to distinguish between different possible outcomes–like for example, if there were 2 or 3 intermediate states, how would that look like as compared to having one (which is what is shown)? I ask because I can imagine this method could be applied also to other proteins as well, which may have more than one intermediate state. Thanks!
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Thank you!
In case of multiple intermediates, you will see multiple minor dips and you can fit them to other models rather than two state model. Also, you can record the experiment at different B1 fields to check if you are missing any hidden or merged minor dips.
I hope that answers the query to some extend!-
Yes it does! Thank you for your response!
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